The present disclosure relates generally to the culture of stem cells. More particularly, the present disclosure relates to cell culture methods for generating colonies of stem cells having controlled size.
The substrate on which cells are cultured is important for successful cellular growth and tissue generation. For example, it has been demonstrated that attachment to the substrate by human embryonic stem cells may contribute to the variability in whether the cells remain undifferentiated or undergo differentiation.
Many protocols for differentiation of pluripotent stem cells begin with the formation of 3-dimensional aggregates of cells called embryoid bodies (EBs). Methods for forming embryoid bodies involve techniques such as scraping adherent ES cell and induced pluripotent stem cell cultures and mild treatment with proteases such as trypsin and/or dispase to release large clumps of cells, followed by placing the resulting aggregates in non-adherent suspension culture. The aggregates formed using these methods are heterogeneous in size and shape, which can lead to inefficient and uncontrolled differentiation. Aggregate size can also directly affect subsequent differentiation pathways. To address these issues, cell culture substrates such as multi-well plates with wells having defined widths have been developed. Another method creates dots of a substrate material such as Matrigel® onto the surface of a plate.
Self-assembled monolayers (“SAMs”) in array formats (i.e., SAM arrays) have been constructed that present ligands to cells plated onto the array. A SAM array is an organized layer of amphiphilic molecules in which one end of the molecule exhibits a specific, reversible affinity for a substrate and the other end of the molecule has a functional group. Because the molecule used to form the SAM array is polarized, the hydrophilic “head groups” assemble together on the substrate, while the hydrophobic tail groups assemble far from the substrate. Areas of close-packed molecules nucleate and grow until the surface of the substrate is covered in a single monolayer. The use of alkanethiols to construct SAM arrays allow for the formation of reproducible SAM arrays and surfaces. SAM arrays may be used to identify specific ligands or epitopes that promote cellular attachment, spreading, proliferation, migration and differentiation, as well as for modulating these cellular activities differentially on each spot on the same SAM array.
Aggregate size and shape can also directly affect subsequent differentiation pathways and lead to inefficient and uncontrolled differentiation. Accordingly, there exists a need for alternative substrates and methods to control the size and/or shape of colonies as well as avoid treatments such as scraping and enzymes used to harvest the cell aggregates.